The growth of Flammulina filiformis is strongly dependent on low-temperature cues for the initiation of primordia formation. To obtain a comprehensive understanding of the molecular mechanisms that govern the mycelial response to cold stress, de novo genome sequencing of the F. filiformis monokaryon and multi-omics data (transcriptome and metabolome) analyses of the mycelia, primordia, and fruiting bodies were conducted in the present study. Genome sequencing based on PacBio HiFi and Hi-C resulted in a 36.3 Mb genome sequence that mapped to 12 chromosomes, comprising 11,886 protein-coding genes. A total of 25 cold-responsive (COR) genes and 520 cold-adapted enzymes were identified in the genome. Multi-omics analyses showed that the pathways related to carbohydrate metabolism in the mycelia under low temperature (10 °C) were significantly enriched. Further examination of the expression profiles of carbohydrate-active enzymes (CAZymes) involved in carbohydrate metabolism revealed that out of 515 CAZyme genes in F. filiformis, 58 were specifically upregulated in mycelia under low-temperature conditions. By contrast, the expression levels of these genes in primordia and fruiting bodies reverted to those prior to low-temperature exposure. These indicate that CAZyme genes are important for the low-temperature adaptation of F. filiformis. This research contributes to the targeted breeding of F. filiformis.

Although gene editing technologies have advanced considerably, research on gene editing in Flammulina filiformis remains in its infancy, and a systematic evaluation of how ssODN donor properties affect HDR efficiency in this species is still lacking. To enable precise genome editing in the edible mushroom F. filiformis, we established an LbCas12a ribonucleoprotein (RNP)-mediated homology-directed repair (HDR) platform in protoplasts and systematically evaluated key ssODN donor design parameters: PAM-site mutation, homology arm distance, RAD51-preferred sequences (RPSs), truncated Cas target sequences (tCTSs), the RS-1 enhancer, and thermodynamic stability (ΔG). A PAM-disrupting mutation greatly increased HDR efficiency, up to 58.5%. Extending the left homology arm 174 bp upstream of the break still yielded 20.2% HDR, expanding the tolerable distance. RPSs showed context-dependent effects: enhancing HDR up to 287.8% in some designs but reducing it by 61.9% in another. Both tCTS and RS-1 consistently suppressed HDR. No strong linear correlation between ΔG and HDR efficiency was observed. Collectively, from experiments conducted at a single genomic locus, our results not only establish a versatile precision-editing platform for F. filiformis through optimized ssODN design, but also offer tentative and transferable insights into the HDR-mediated genome editing of other heterokaryotic basidiomycetes facing similar genetic operation challenges.